DETECTION OF BIOLOGICAL WARFARE AGENTS. Aaron King G. Dela Peña

Transcription

1 DETECTION OF BIOLOGICAL WARFARE AGENTS Aaron King G. Dela Peña

2 Outline Introduction Goals Detection Methods Conclusion

3 Introduction Biological warfare agents (BWAs) are toxins of biological origins or microorganisms used as weapons of mass destruction BWAs can include bacteria, viruses, and fungi Requires incubation period before victim shows any serious symptoms Can be passed on from person to person Advances in molecular biology and genetic engineering, magnifies the threat of BWAs 1. Ivnitski, D., O Neil, D.J., Gattuso, A., Schlicht, R., Calidonna, M., and Fisher, Rodney. Nucleic acid approaches for detection and identification of biological warfare and infectious disease agents. Rapid Detection and Identification of Infectious Disease Agents, 2003, 35, p Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

4 Introduction cont d Qualities for a good biological warfare agent (BWA) are relationship between aerosolization, infectivity, or toxicity and the concentration required to produce an effect Qualities may also include environmental stability, ease of production, disease severity Highly lethal and limited options for preventive treatment (ie respiratory route most effective for BWA) When testing potential agents for these characteristics Bacuillus anthracis (anthrax) and variola major virus (smallpox) are considered due to potential for large mass casualties and civil disruption May also consider botulinum neurotoxins, Yersinia pestis, and Francisella tularensis 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

5 Goals Accurately predict the dispersion, concentration, and the fate of BWAs in real time Be able to define the perimeter of attack and moving fronts Moved into field or area of attack to provide rapid diagnosis and monitoring Ability to discriminate between closely related organisms Provide threshold concentrations at minimum five to ten minutes 1. Ivnitski, D., O Neil, D.J., Gattuso, A., Schlicht, R., Calidonna, M., and Fisher, Rodney. Nucleic acid approaches for detection and identification of biological warfare and infectious disease agents. Rapid Detection and Identification of Infectious Disease Agents, 2003, 35, p 1-8.

6 Detection Methods Immunochromatographic lateral flow assays (handheld device assays) Enzyme-linked immunosorbent assay (ELISA) Time-resolved Fluorescence (TRF) Bead Array Counter (BARC)

7 Immunochromatographic Lateral Flow Assays First described in late 1960s and was originally developed for assessment of the presence of serum protein Over the past decades, used for detection for infectious diseases, cancer, cardiovascular problems, pancreatitis and illicit drugs Other potential uses include drug monitoring, food safety, and veterinary medicine 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

8 Immunochromatographic Lateral Flow Assays cont d Contain colloidal gold-labeled antibody dried onto a filter pad affixed to a nitrocellulose strip Capture antibody applied on strip and dried To perform a test: Sample suspended in a buffer and placed on the pad Antibody w/ gold binds specifically to the antigen in the sample This antibody-antigen complex binds to a capture antibody A positive reaction is visualize as a red line which is created by bound colloidal gold Takes about 15 minutes 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

9 Improvement on handheld assay devices Limitations with present generation Only one agent can be detected per assay strip Different sensitivity per respective target agents Most: Bacteria ( to CFU/mL) Least: viruses ( to PFU/mL) Visualization (red line): sensitivity limited to what can be seen with unaided eye Improvement Sensitivity can be improved up to one order of magnitude by using a silver enhancement step (eg. ng/ml to pg/ml) 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

10 Enzyme-linked immunosorbent assay (ELISA) Principal Labelling by chemical conjugation of an enzyme bound to either an antigen or antibody Detection of immune complexes formed on solid phase Washed free of excess reagents and subsequent substrate interaction Detection via colored product Economical, versatile, robust, and separation based on bound or free moieties by use of a solid-phase support Can screen large numbers of small-volume test samples 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

11 ELISA cont d For detection of BW/ID agents Capture antibody affixed to solid support Exposed to test samples, including positive and negative control samples Washing occurs to remove any unbound molecules not specifically bound to an antibody The capture antibody-antigen complex is exposed to a detector antibody selective to the antigen in the complex 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

12 Improvements for ELISA The use of monoclonal (from single cell lineage) antibodies usually results in high specificity and low background Potential improvements: could use polyclonal (from different cell lineage) antibodies Increases the breadth of the assay to detect multiple isolates of the same species of bacteria, virus, fungus Can be modified to detect serum immune complexes Antibody isotypes can be determined and quantified by using a panel of isotype-specific antiglobulin conjugates on the same test samples in repeated assays 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

13 Time-resolved fluorescence (TRF) Use of lanthanide chelate labels Key feature in fluorescence properties of these labels include very long fluorescence decay time and exceptionally large Stokes shift Allows for user to measure fluorescence after background has subsided. Contributes to high sensitivity and low background which is characteristic of immunologic assays based on TRF 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

14 Time-resolved fluorescence (TRF) Assays set up in traditional 96-well two-site antigencapture ELISA format Capture antibody on solid support is exposed to sample and washed unbound antigen Primary antibody-antigen complex is then exposed a secondary antibody with a lanthanide label (usually Europium, Eu 3+ ) Acidic enhancement solution is added to have the label dissociate which is highly fluorescent. 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

15 Improvements for TRF Limitations of TRF are similar to ELISA, which primarily have to do with the functions of the antibodies Must perform the experiment well to reduce chance of lanthanide contamination 1. Peruski, A.H. and Peruski, L.F. Immunological Methods for Detection and Identification of Infectious Disease and Biological Warfare Agents. Clinical and Diagnostic Laboratory Immunology, 2003, 4, p

16 Bead ARray Counter (BARC) Being developed to use DNA hybridization, magnetic microbeads and giant magnetoresistive (GMR) sensors for detection of biomolecules Has the ability to discriminate between the species that are specifically and non-specifically bound using a magnetic field The applied force is used to test for the bonding strength between the interacting molecules GMR materials generally thin-film multilayers that can have its resistance changed in response to a magnetic field Measures local magnetic field Used in BARC sensors: detects presence of magnetic microbeads which was developed for separations and purifications Edelstein, R.L., Tamanaha, C.R., Sheehan, P.E., Miller, M.M., Baselt, D.R., Whitman, L.J., and Colton, R.J. The BARC biosensor applied to the detection of biological warfare agents. Biosensors & Bioelectronics, 2000, 14, p

17 BARC instrument overview Table top instrument with: Microfabricated chip with an array of GMR sensors A chip carrier board with electronics for lock-in detection A fluidics cell and cartridge An electromagnet Edelstein, R.L., Tamanaha, C.R., Sheehan, P.E., Miller, M.M., Baselt, D.R., Whitman, L.J., and Colton, R.J. The BARC biosensor applied to the detection of biological warfare agents. Biosensors & Bioelectronics, 2000, 14, p

18 BARC Experiment Sample preparation DNA sample labeled with biotin during polymerase chain reaction (PCR) amplification Labeled sample injected, will hybridize with probes if there are complementary sequence on the chip surface Streptavidin covered paramagnetic beads added that will bind to biotinylated DNA sample Magnetic field applied, removes any unbound beads GMR detects the remaining beads, concentration of pathogen in sample determined by the intensity and location of the signal Samples used: Bacillus anthracis, Yersinia pestis, Brucella suis, Francisella tularensis, Vibrio cholerae, Clostridium botulinum, Campylobacter jejuni, and Vaccinia virus Edelstein, R.L., Tamanaha, C.R., Sheehan, P.E., Miller, M.M., Baselt, D.R., Whitman, L.J., and Colton, R.J. The BARC biosensor applied to the detection of biological warfare agents. Biosensors & Bioelectronics, 2000, 14, p

19 BARC experiment cont d Gold substrates arrayed with a DNA probe for B. anthracis lethal factor Top panel: Gold substrate exposed to non-complementary biotinylated DNA, nonspecific adhesion of DNA and of the magnetic microbeads is very low; >95% of beads removed when magnetic field applied Bottom panel: specific hybridization of single-stranded complementary oligonucleotide to the arrayed area on the substrate The ability to do more than one assay at a time Three different probes arrayed across the gold substrates Thiolated and biotinylated probe on top row (control) B. anthracis lethal factor (ALF) probe on second row C. botulinum neurotoxin A (BA) probe third row Edelstein, R.L., Tamanaha, C.R., Sheehan, P.E., Miller, M.M., Baselt, D.R., Whitman, L.J., and Colton, R.J. The BARC biosensor applied to the detection of biological warfare agents. Biosensors & Bioelectronics, 2000, 14, p

20 Conclusion Advances in immunological reagents and assay formats are being matched with advances with laboratory technology These advances are offset by the fact that the size and delicate nature of the current systems are limited to the laboratory.